BRPI9916095B1 - cell and cell line expressing a heterologous protein - Google Patents

Abstract

"Human hybrid host cell for mammalian gene expression". hybrid / human / human cells are made via fusion of human embryonic kidney cells (293s) and modified brukitt lymphoma cells (2b8). fusion cells are usable as host cells for recombinant expression of mammalian genes. The advantages of using these hybrid human kidney b and b clones, called hkbs, for mammalian gene expression, include (i) the cells are negative for immunoglobulin expression, (ii) the cells grow easily in protein free medium. plasma, (with or without the addition of recombinant insulin) as suspension cultures in a shake flask or fermenter (iii) cells are very susceptible to DNA transfection, and (iv) cells secrete high levels of proteins. heterologous recombinants, such as recombinant monoclonal antibodies, soluble icam-1, ril-4 and rfviii.

Description

Invention Patent Descriptive Report for "CELL AND CELL LINING EXPRESSING A HETEROL-GA PROTEIN".

Related Requests: The order for Cho and Chan designated MSB-7254 (U.S. Serial Number 09 / 209,915), "Vectors Having Terminal Repeat Sequence of Epstein-Barr Virus," and the request for Cho et al. designated MSB-7255 (U.S. Serial Number 09 / 209,916), "Expression system for factor VIII," contains related subject matter. Both applications were filed on December 10, 1998.

BACKGROUND OF THE INVENTION

Field: This invention generally relates to genetically engineered mammalian cell lines for the production of biologically active protein. Specifically, the invention relates to human hybrid cell clones derived from the human embryonic kidney (293S) cell fusion process and Burkitt's lymphoma cells. These human hybrid cells can be used for the production of heterologous proteins.

Background: Until now, most therapeutic recombinant proteins have been produced from non-human mammalian cells. Some examples are: Chinese Hamster Ovary Cell (CHO) (dhfr-) (Urlaub et al., 1980, Proc Natl Acad Sci USA 77: 4216 - 4220) with amplifiable selection marker dihydrofolate reductase. (Kaufman et al., 1982, Mol. Biol. 159: 601-621; Gasser et al., 1982, Proc Natl Acad Sci USA 79: 6522-6526) have been used for the production of therapeutic recombinant protein. Various recombinant therapeutic proteins are known to be produced in mammalian cells, for example recombinant factor VIII (rFVIII) (Kaufman et al., 1988, J Biol Chem 263: 6352 - 6362), tissue plasminogen activator (tPA) (US patent). 4,766,075 to Goeddel et al., 1988), erythropoietin (EPO) (US patent 4,703,008 to Lin, 1987), and monoclonal antibodies (mAbs) (US patent 4,816,397 to Boss et al., 1989).

BHK baby hamster kidney (BHK21) cells were used for rFVIII production after G418 selection and methotrexate (MTX) amplification of G418 resistant cells (U.S. Patent 4,965,199 to Capon et al., 1990).

Mouse myeloma (NSO) cells were used to produce engineered human anti-TNF antibody (EHAT) (U.S. Patent 4,816,397 to Boss et al., 1989). However, this cell line produces proteins having mouse-specific carbohydrate patterns that are not favorable for human use.

One human cell line, Namalwa (from Brukitt's lympho-ma) was used for alpha-interferon production by the Wellcome Research Laboratory, and for the production of pro-urokinase (Satoh et al., 1996, Cytotechnology 18: 167 - 185, 1996), plasminogen-tissue activator (t-PA) (Khan et al., 1995, Biochem Soc Trans 23: S99), granulocyte-macrophage colony stimulating factor (Okamoto et al., 1991, Arch Biochem Bio-phys 286: 562 - 568), interferons and lymphotoxin (Hosoi et al., 1991, Cytotechnology 5: 17 - 34), and granulocyte colony stimulating factor (Hosoi et al., 1991, Cytotechnology 7: 25 - 32 ) by Tokyo Research Laboratories. However, these cells were very difficult to transfect with DNA.

Walls et al. (1989, Gene 81: 139 - 149) recorded the use of dhfr / MTX co-amplification strategy to express functional protein C in human embryonic kidney cells (293S cells). 293 cells (Stillman et al., 1985, Mol. Cell. Biol. 5: 2051 - 2060) are known to make large suspended aggregates, especially under high calcium concentration (> 100 μΜ), which promotes greater aggregation and lower viability. of the cell (Peshwa et al., 1993, Biotech and Bioeng 41: 179 - 187). All references cited are incorporated herein by reference.

SUMMARY OF THE INVENTION

Hybridized human cell clones have now been established which are easily transfected by electroporation or cationic liposome and which are easily adapted for growth in suspension culture. Heterologous proteins can be expressed using low levels (50-100 nM) of MTX amplification in a human cell medium. In addition, cells are easily adapted for growth in serum free medium.

These cells are the product of a fusion between human embryonic kidney cells (293S) and Burkitt lymphoma cells. See figure 1 for a summary. These hybrid clones, called HKBs, harbor a defective EBV genome derived from HH514-16, which is a cell line originating from Burkitt lymphoma cells, P3HR1 (Hinuma et al., 1967, J Virol 1: 1045 - 1051). . P3HR1 cells harbor non-immortalizing EBV. HH514-16 is a P3HR1 clone (Hinuma et al., 1967, J Virol 1: 1045-1051) which houses non-immortalizing EBV. HH514-16 lost a het-DNA, a latency disrupting DNA, during the cloning process (Rabson et al., 1983, Proc Natl Acad Sci USA 87: 3660-3664). Thus, HVB clone EBV is a non-immortalizing virus and remains a latency. HKBs are human hybrid host cells suitable for recombinant production of therapeutic proteins. These host cells are obtained by hybridizing to different parent cell lines, each having different advantageous characteristics. Host cells which have advantageous characteristics of each of the parental cell lines are obtained from the resulting hybridization cells.

Host cells can be genetically engineered to express high levels of a wide range of proteins. Proteins that can be produced by engineered host cells include, but are not limited to, soluble ICAM-1, recombinant interleukin -4, (IL-4), tPA, EPO, rFVIII, and BDD-FVIII (deleted domain B variants of factor VIII), and derivatives of these proteins. Factor VIII has an A1-A2-B-A3-C1-C2 domain organization and is synthesized as a 2351 amino acid single-stranded polypeptide, of which a 19 amino acid signal peptide is cleaved upon translocation in the reticulum lumen. endoplasmic. Due to the fact that factor VIII is strongly glycosylated, high level expression (> 0.2 pg / c / d) of factor VIII was difficult to obtain (Lind et al., 1995, Eur J Biochem 232: 19 - 27 Kaufman et al., 1989, Mol Cell Biol. 9: 1233-1242). Factor VIII expression in mammalian cells is typically 2-3 orders of magnitude lower than that observed with other genes using similar vectors and approaches. Productivity of production cell lines for factor VIII was in the range 0.5-1 U / c / d (0.1-0.2 pg / c / d).

Factor VIII domain B has been shown to be dispensable for procoagulant activity. Using truncated factor VIII variants, improved factor VIII expression in mammalian cells has been recorded by several groups (Lind et al., 1995, Eur J Biochem 232: 19 - 27; Tajima et al., 1990, Proc 6th Int Symp Η T. 51-63; US Patent 5,661,008 to Almstedt 1997). However, the expression level of factor VIII variants remained below 1 pg / c / d of a stable cell clone. It was also found that although endogenous immunoglobulin (Ig) was not expressed, expression of recombinant engineered host cell Ig was high. The proteins produced from HKB11 cells have human specific glycosylation profile. Thus, clones are great host cells for the production of engineered Ig by genes and other proteins.

As used herein, a Burkitt lymphoma origin cell is a cell that is a Burkitt lymphoma cell, derived from a Burkitt lymphoma cell, derived from another Burkitt lymphoma origin cell, or a cell resulting from division mitotic of any of the above. "Derivatives of" in this context are intended to include, but are not limited to, normal mitotic cell division, and processes such as transfections, cell fusions, or other cell biology or genetic engineering techniques used to alter cells or produce cells with New properties. Similarly, a human embryonic kidney origin cell is a cell that is a human embryonic kidney cell, derived from a human embryonic kidney cell, derived from another human embryonic kidney origin cell, or a cell resulting from the mitotic division of any of the above. Also, a source cell in 293S is a cell that is a cell 293S, derived from cell 293S, derived from another source cell in 293S, or a cell resulting from mitotic division of any of the above. Also a source cell in 2B8 is a cell that is a 2B8 cell, derived from a 2B8 cell, derived from another source cell in 2B8, or a cell resulting from mitotic division of any of the above. A heterologous protein is a protein that a cell has been engineered to produce.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a summary of HKB cell derivation.

Figure 2 shows physical maps of expression vectors mentioned in the text. All plasmids were constructed based on a pBR322 dorsal structure and contain a dhfr expression unit. All genes encoding proteins of interest are under CMV enhancer / promoter regulation (CMVe / p), 5'intron (MIS or CIS) was positioned at the 5 'end of the genes except BZLF1. Poly A signal region was indicated as pA. Both plasmids, pSH157 and pCIS25DTR, contain an EBV-TR (402 bp) sequence. Figure 3 shows a comparison of host cell lines for gene expression in transient transfection assay. Transfections were performed under the same conditions, the same number of cells from 293S, 2B8, and HKB11 were transfected with the same amount of plasmid DNAs using the same transfection agent. Tissue culture fluids were collected 2 days after transfection. Protein production levels were determined by ELISA (IgG and ICAM-1; measured as ng protein / 106 cells / 2 days), and by Coatest® Assay Kit (rFVIII; measured as milliunits / 107 cells / 2 days). SPECIFIC EMBODIMENTS Materials and methods HH514-16 was kindly provided by Dr. George Miller (Yale University). 293S cells were obtained from Dr. Brad Zerler (Molecular Therapeutic Institute, West Heaven, CT). 293S cells are 293 cells (ATCC CRL 1573) that have been adapted to grow in suspension culture (Stillman et al., 1985, Mol Cell Biol 5: 2051-2060).

Plasmids All expression vectors used in this report are primarily pBR322-based plasmid with dhfr gene expression segment function. Physical maps of expression vectors are depicted in Figure 2. Plasmids pSH157 and pCIS25DTR also have Epstein-Barr virus terminal repeat sequence (EBV-TR). See patent application for Cho and Chan designated MSB-7254, "Terminal repeat sequence of Epstein-Barr virus drug selection ratio", for the EBV-TR sequence. The pSH131 vector, which has been deposited with the American Type Culture Collection, ATCC 98879, can be used to generate expression vectors for a chosen protein as described in Cho and Chan (MSB-7254, supra.) ELISA

To measure a tlCAM-1 secretion, a monoclonal antibody to ICAM-1, C92.5 (McClelland et al., 1991, Proc Natl Acad Sci USA 88: 7993-7997) was adsorbed onto round bottom microtiter plates. The plates were blocked by treatment with a phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) then incubated with samples containing tlCAM-1. The plates were then washed with wash buffer (PBS plus 0.005% Tween 20) and incubated with biotinylated C78.5, a second monoclonal antibody for a different t1CAM-1 epitope (McClelland et al., 1991, supra). After washing, the plates were incubated with HRP-streptavidin. The plates were then washed with wash buffer, reacted with tetramethyl benzidine (TMB) and quenched with 1 N HCL. The concentration of tlCAM-1 was determined by reading OD at 450/750 nm and comparing it to a purified tlCAM-1 standard curve.

To measure immunoglobulin (Ig) secretion, anti-1g antibody was used to coat the plate and biotinylated anti-1g antibody was used as a detection antibody. A known concentration of Ig molecule was used as standard. Otherwise, the process remained the same.

To measure interleukin 4 (IL-4) secretion, anti-IL-4 antibody was used to coat the plate, and biotinylated anti-IL antibody was used as the detection antibody. A purified IL-4 molecule was used as standard. Assay for Measuring rFVIII Secretion The rFVIII molecule was quantified with Coatest® VIII: C / 4 kit reagents (Chromogenix, Mölndal, Sweden). A US standard antihemophilic factor (factor VIII) known as MEGA 1) (Office of Biological Research and Re-view, Bethesda, MD) was used as the measurement standard for EIA / RIA A / 2 plates (Corning, Corning, NY) preheated to 37 ° C in Select Heat Blocks (VWR Scientific, San Francisco, CA). Factor IXa, factor X, phospholipid and CaCb were added to each sample and incubated for 10 min for factor X activation. A chromogenic substrate (S2222) was then added and incubated for 10 min to release the chromogenic pNA group. This reaction was stopped by the addition of 50% acetic acid. Colorimetric absorbance was then measured at 405/450 nm on a SPECTRAmax® 250 spectrophotometer microplate reader (Molecular Devices, Sunnyvale, CA) and data calculated via SOFTmax® software provided by Molecular Devices.

H4-Sensitive G418 Burkitt Lymphoma Cell Line Derivation

To obtain HAT-sensitive cells, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient cell lines (Szybalska et al., Proc. Nat. Acad. Sci. USA 48: 2026 - 2034, 1962; Littlefield, Proc. Natl. Acad Sci. USA 50: 568 - 576, 1963) were established by the standard protocol described by Siadak et al. (U.S. Patent 4,834,975, 1989).

HH514-16 cells (obtained from Dr. G. Miller, Yale University), which are free of EBV het-DNA (Rabson et al., 1983, Proc Natl Acad Sci USA 87: 3660 - 3664), were treated with 300 µg. / ml methanesulfonic acid (MSE) ethyl ester (Sigma, St. Louis, MO) in RPMI-1640 suspension medium (Life Science, Gaithersburg, MD) supplemented with 15% fetal bovine serum (FBS) (Hyclone, Logan, UT) for 24 hours. After washing the cells with medium, cells were placed in medium containing 6-thioguanine (6TG) (Sigma) (5 pg / ml) to select for HGPRT negative cells. The 6TG concentration was increased from 5 μ9 / ιτιΙ to 30 pg / ml during the six month selection period. The cells were then tested for their sensitivity to HAT-containing medium. Single cell clones (SCCs) were obtained from dilution cloning limitation (one cell per well in 96 well plates) of HAT A5 sensitive population. One of the SCCs, A5 / 1D7 was transfected with pSV2neo, which has a neo gene under p40 vector SV40 promoter, to obtain G418 resistant cells. One of the G418 resistant SCCs (1.5 mg / ml) referred to as 2B8 (deposited with American Type Culture Collection, No. CRL-12569) was used for fusion. EXAMPLE 1 Cell Fusion and Derivation of Single Cell Clones Cell fusion was primarily performed according to the polyethylene glycol (PEG) fusion process described by Kennett (1979, Meth Enzymol 58: 5 - 359). Five million logarithmic 293S and 2B8 cells each were washed with PBS without Ca ++ and Mg ++ (Life Technologies, Rockville, MD) and seeded into a reservoir of a peanut agglutinin pretreated 6-well plate (Sigma) (5 pg / ml). Cell loaded 6-well plates were centrifuged at 400g for 6 min in a Beckman J-6M / E centrifuge (Beckman, Palo Alto, CA). After removing PBS from the reservoir, cells were treated with 2 ml of 40% (w / v) PEG (Sigma) for one minute). As a control, a reservoir was not treated with PEG. Cells were washed three times with 5 ml PBS containing 5% DMSO followed by three PBS wash. Cells were incubated with fresh medium supplemented with 15% FBS for 25 min. Cells were seeded in 96 well plates (1.2 x 10 6 cells per plate) using fresh medium containing G418 (1 mg / ml) and HAT) (Life Technology) supplemented with 15% FBS. The cells were fed twice a week using a selection medium. In this example, for initial selection, 293S cells had the desirable characteristic of lacking sensitivity to the HAT-containing medium, and similarly, 2B8 cells had the desirable characteristic of being resistant to G418.

Although fused cells grow under selective conditions, mixed cells do not grow under the same conditions. Three weeks after selection, initial populations were transferred to larger formats. To obtain SCCs, the twenty initial stable growth populations were mixed and cloned by limiting dilution (one cell per reservoir) using the selective medium. 19 SCCs were selected from 15 x 96 well plates after careful monitoring of individual clones using a microscope. SCCs derived from the fusion experiment were reported with HKB cells (B-cell and human kidney hybrid cells). Seven SCCs were further tested for stable production of various proteins. One of seven SCCs, HKB11, was chosen as a preferred mammalian cell host for the production of heterologous proteins. See figure 1 for a summary. EXAMPLE 2 Characterization of HKB Clones

Although all hybrid cells were selected under selective conditions, the hybrid state was confirmed by counting chromosome numbers in cells. In fact, all HKBs showed a modal chromosome number of 90-110. These numbers are similar to the sum of modal chromosome numbers of 293S and 2B8 (64 and 47, respectively, according to ATCC data). 293S (Stillman et al., 1985, Mol Cell Biol 5: 2051-2060) 293 suspension adapted (ATCC CRL-1573).

All hybrid cell clones were tested for endogenous Ig expression (mu and Kappa) by direct immunofluorescence testing using methanol-fixed cells to determine which endogenous Ig secreted cell clones. In repeated experiments over a longer period, these cells were observed to be negative for expression of mu- and kappa chains based on immunofluorescence assay (table 1) and ELISA (data not shown).

Table 1. Detection of Ig gene expression.

Gene Expression Cells _____________________ heavy chain (mu) light chain (kappa) 293S Negative Negative 2B8 Positive HKB Negative Negative Table 1 shows the observed results of the three cell type immunofluorescence test. The cells were resuspended in PBS and applied as a glass slide smear. After drying the cells, the slides were fixed in cold (-20 ° C) methanol for 5 min. Cells were stained with anti-human FITC kappa and anti-human mu strands (1:20 dilution) (Zymed Laboratories, Inc., San Francisco, CA) in a humidified chamber at 37 ° C for 45 min. After rinsing slides with PBS for 10 min, slides were mounted with cover glass using PBS / glycerol (1: 1). Cells were observed under a fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY). Human specific sialic acid binding pattern, alpha (2-6) sialyltransferase was confirmed by FACS analysis of HKB cells using FITC-conjugated Sambucus nigra lectin (SNA) (Sigma, St. Louis, MO). HKB cell secreted protein (clone 1G2) showed alpha (2-3) and alpha (2-6) binding of glycosylation profile sialic acid analyzed by oligosaccharide fingerprinting process (data not shown). This observation indicates that HKB cells derived from fusion of 293S and 2B8 cells maintained human specific glycosylation enzymes.

To test whether these hybrid cells have the ability to express foreign genes, the cells were transfected with expression vectors for ICAM-1 and IgG (anti-TNF antibody). To test for IgG secretion, HKB cells (5 x 106 cells) were transfected by electroporation with 10 μg of plasmid DNA providing functional expression of heavy (gamma) and light (Kappa) chains. To test soluble ICAM-1 secretion levels, HKB cells (5 x 10 6 cells) were transfected by electroporation with 10 µg of plasmid DNA providing functional expression of soluble ICAM-1. Secretion levels were determined by an IgG or ICAM-1 ELISA. All 19 SCCs secreted relatively high levels of ICAM-1 (100 - 500 ng / ml / 2d) and IgG (30 - 200 ng / ml / 2d) in a transient transfection assay (Table 2). These data indicate that hybrid cells support expression of transfected Ig genes, although endogenous Ig gene expression is extinct. Epstein-Barr virus (EBV) exists as episomes in 2B8 cells. All SCCS were positive for EBNA-1 expression (data not shown), indicating that they are EBV positive. However, it is not known whether a complete EBV genome still existed in hybrid cells.

Table 2 - Transient transfection assays for soluble IgG and ICAM secretion of HKB clones that are obtained prematurely after the cloning process. Values are protein secretion levels (Ng / ml / 2d).

Nd = not performed Thus, the state of the EBV genome in hybrid cells was tested by transfecting cells with the EBV genome fragment BZLF1, a transactant latency disruption gene. HKB cells (5 x 10 6 cells) were transfected with 10 pG plasmid DNA (pSH121) allowing functional expression of BZLF1. Detection of EBV capsid antigen (EBV-VCA) was performed by indirect immunofluorescence using human serum containing anti EBV-VCA titer and FITC-conjugated anti-human IgG. The technical details of immunofluorescence testing are described above. As in Table 3, cells transfected as imitation were negative for EBV capsid antigen (EBV-VCA) expression, which indicated that they were negative for EBV replication. However, a small percentage of transfected cells were positive for antigen expression. These data indicate that the hybrid cells harbor the latent EBV genome.

Table 3 - Induction of EBV replication by transfection with BZLF1.

Hybrid cells were adapted to serum free medium by double serial reduction of FBS in shake flasks. After two weeks, the cells were growing in medium without FBS. Cells were grown as small aggregates in shake flasks. In contrast to 293S cells, hybrid cells were easily adaptable to serum-free suspension cultures. Hybrid cells in serum-free and albumin-free medium supplemented with transferrin and insulin may be maintained for more than one year in suspension culture using shake flasks.

To compare levels of secretion of transfected gene products, one of the clones, HKB11 and parental cells 293S and 2B8, were transfected with pSM98 (ICAM-1 soluble), pSH125 (anti-TNF IgG), and pCISF8 (rFVIII). . As shown in Figure 3, ICAM-1 and IgG secretion levels were much higher (approximately 10-fold) in HKB11 cells than 293S cells. Secretion levels for both 2B8 proteins were not detectable. The levels of rFVIII secretion in HKB11 cells were similar to those of 293S cells. These data indicate that the transfection efficiency of HKB11 cells were lower than parental cells. EXAMPLE 3 Derivation of stable HKB clones secreting heterologous proteins Hybrid clones were tested for stable protein expression in an amplifiable gene system (dhfr / MTX). Cells were first transfected with an appropriate expression vector, and then transfected cells (usually 106 cells per 96 well plate) were seeded and screened / amplified with the missing hypoxanthine and thymidine selection medium, but supplemented with FBS and MTX (50 nM). After the first plate screening, the high secretion reservoir cells were picked and transferred to the 6 reservoir plates. Cells in 6 well plates were further amplified with increasing concentrations of MTX (100, 200, and 400 nM) in medium. Initial plate populations of 6 reservoirs were further screened to derive the best population. Eventually, these populations were used for cloning single cell derived clones. As shown in table 4, HKB11 cells were optimal for producing high levels of heterologous human proteins. For the production of ICAM-1, Ig, and an IL-4 derivative, HKB11 cells were as good as CHO cells (data not shown). However, HKB clones grow faster than CHO clones and can be easily adapted to suspension culture and serum free conditions.

In the case of BDD-FVIII production, HKB11 clones showed about ten times higher productivity than CHO clones. The above results indicate that HKB11 cells are an optimal human cell host usable for expression of human therapeutic proteins.

Table 4 - HKB Clone Heterologous Protein Production

Protein Host Cell MTX Amplification Specific Productivity ICAM-1 HKB11 100 nM 10pg / c / d1) Ig HKB13 50 nM 12pg / c / d BDD-FVIII HKB11 400 nM 5-10uU / c / d2) IL-4SA _____________ HKB11___________100 nM_______________5 pg d 3) _______ 1) ICAM-1 production level of HKB clones was similar to that of 293S cells. Clone HKB secreting ICAM-1 was easily adaptable to suspension culture, while 293S clones were very difficult to adapt to suspension culture. 2) the level of BDD-FVIII secretion was about 10-fold higher than that of clones derived from CHO cells transfected with the same expression vector. 3) gram amount of IL-4SA production (T13D / R121E) was possible 6 months after transfection using HKB11, while it took a few more months using CHO cells in simultaneous experiments using the same expression vector and similar process.

Cell lines derived from the above process included (i) soluble ICAM-1 secreting clones (10 pg / cell / day) derived from pSM98 transfected HKB11 cells after amplification at 100 nM MTX, (ii) monoclonal antibody (anti-TNF) secreting single cell clones (12 pg / cell / day) derived from pSS125 transfected HKB13 after amplification by 50 nM MTX and limiting dilution cloning without MTX, (iii) truncated rFVIII (BDD-FVIII) secreting single cell clones ( 5-10 μυ / c / d, in serum-free condition), derived from pCIS25DTR-transfected HKB11 cells after amplification (400 nM MTX) and limiting dilution cloning without MTX, and (iv) IL-4 derivative (selective for agonist). IL-4, IL-4SA, two amino acid shifted positions, T13D and R121E) secreting (5 pg / c / d) clones derived from pSH157 transfected HKB11 after ampl amplification. Clone secreting IL-4SA 1G2 was used for relatively rapid production of small amounts of protein (gram amount). See Figure 2 for the physical map of expression vectors mentioned above.

DISCUSSION

The hybrid human cell lines herein show desirable characteristics possessed by their parent cell lines. HKB cell lines, which are produced by fusion of human embryonic kidney cells (or cells derived from human embryonic kidney cells) with Burkitt lymphoma cells (or cells derived from Burkitt lymphoma cells) are usable for development. of cell lines for the expression of heterologous proteins. The initial purpose of establishing the 293S and 2B8 hybrid cell lines was to solve the 293S cell aggregation problem, which tends to cluster when grown in suspension culture (an undesirable feature). HKB cells that were developed from the hybridization process grow as a monolayer when grown in T-flasks. However, these cells grow as suspension cells (do not form large aggregates) when they are cultured in suspension mode. HKB cells are very easy to manipulate for transfection, and transfection efficiency is much higher than with 293S cells.

Although the transformation or hybridization event required in most cases to produce a stable cell line results in altered glycosylation profiles (Yamashita, 1989, J Biol Chem 264: 2415 - 2423), it has been found that HKB11, which is a Somatic hybrid cell line has typical human glycosylation enzymes, for example alpha (2-3) and alpha (2-6) sialyl transferases. In addition, proteins produced from transfected HKB cells have normal human glycosylation patterns of alpha (2-3) and alpha (2-6) sialic acid bonds.

In summary, HKBs are hybrid human cells having desirable characteristics of each parent cell line, that is, they grow easily in suspension culture without aggregation (as observed with 2B8 cells). The preferred hybrid human cell line, HKB11, is negative for immunoglobulin gene expression as are 293S cells. This feature is advantageous in cases where it is desired to recombinantly produce monoclonal antibodies from human cells. The finding that fusion of human cells has resulted in cells having advantageous characteristics serves to encourage further fusion studies using 293S and other B-cell origin strains, for example Na-malwa and 6F11, to obtain new trait combinations in the cells. hybrid fusion cells of different parental cell lines.

The above examples are intended to illustrate the invention and it is believed that variations will occur to those skilled in the art. Accordingly, it is intended that the scope of the invention be limited only by the claims below.

Claims (14)

1. Cell, characterized in that it is designated by the American Type Culture Collection (ATCC) as CRL-12568 and is derived from the fusion of a 293 cell with a 2B8 cell (ATCC filing number: CRL-12569). Cell according to claim 1, characterized in that it expresses a heterologous protein. Cell according to claim 2, characterized in that the heterologous protein is selected from the group consisting of FVIII, BDD-FVIII, monoclonal antibody, anti-TNF antibody, rLL4, tPA, and E-PO. 4. Cell line expressing a heterologous protein, characterized in that the cell line derived from the cell line is designated by the American Type Culture Collection (ATCC) as CRL-12568. Cell line according to claim 4, characterized in that the protein is ICAM-1. Cell line according to claim 4, characterized in that the protein is BDD-FVIII. Cell line according to claim 4, characterized in that the protein is a monoclonal antibody. Cell line according to claim 7, characterized in that the monoclonal antibody is anti-TNF. Cell line according to claim 4, characterized in that the protein is rLL4. Cell line according to claim 4, characterized in that the protein is FVIII. Cell line according to claim 4, characterized in that the protein is tPA. Cell line according to claim 4, characterized in that the protein is EPO. Cell line according to claim 4, characterized in that the protein is IL-4SA and the cell line is designated by the American Type Culture Collection (ATCC) as PTA-87. Cell line according to claim 4, characterized in that the protein is a human protein having a human glycosylation profile.